Methods for determining 2F1-binding compounds

ABSTRACT

2F1 polypeptides are provided, along with DNA sequences, expression vectors and transformed host cells useful in producing the polypeptides. Soluble 2F1 polypeptides find use in inhibiting prostaglandin synthesis and treating inflammation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.10/857,664, filed May 28, 2004, now issued as U.S. Pat. No. 7,235,238,which is a Continuation of U.S. patent application Ser. No. 10/157,447,filed May 28, 2002, now issued as U.S. Pat. No. 7,001,599, which is aContinuation of U.S. patent application Ser. No. 09/578,178, filed May24, 2000, now issued as U.S. Pat. No. 6,451,760, which is a Divisionalof U.S. patent application Ser. No. 09/110,618, filed Jul. 6, 1998, nowissued as U.S. Pat. No. 6,090,918, which is a Divisional of U.S. patentapplication Ser. No. 08/604,333, filed Feb. 21, 1996, now issued as U.S.Pat. No. 5,776,731, which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The type I interleukin-1 receptor (IL-1RI) mediates the biologicaleffects of interleukin-1, a pro-inflammatory cytokine (Sims et al.,Science 241:585-589, 1988; Curtis et al., Proc. Natl. Acad. Sci. USA86:3045-3049, 1989). A second interleukin-1 receptor (designated type IIIL-1R or IL-1RII) binds IL-1, but does not appear to mediate signaltransduction (McMahan et al., EMBO J. 10:2821, 1991; Sims et al., Proc.Natl. Acad. Sci. USA 90:6155-6159, 1993). IL-1RI and IL-1RII each bindIL-1α and IL-1β.

IL-1RI and IL-1RII belong to a family of proteins that exhibitsignificant sequence homology. One such protein is IL-1R accessoryprotein (IL-1R AcP), described in Greenfeder et al. (J. Biol. Chem. 270:13757-13765, 1995). This protein, by itself, is not capable of bindingIL-1, but does form a complex with IL-1RI and either IL-1α and IL-1β.When co-expressed with IL-1RI, recombinant IL-1R AcP increases thebinding affinity of IL-1RI for IL-1β (Greenfeder et al., supra).

The protein variously known as ST2, ST2L, T1, or Fit-1 also is a memberof the IL-1R family, but does not bind IL-1. Cloning of mouse and ratcDNAs encoding membrane-bound and secreted forms of this protein hasbeen reported (Klemenz et al., Proc. Natl. Acad. Sci. USA 86:5708, 1989;Tominaga, FEBS LETTERS 258:301, 1989; Yanagisawa et al., FEBS LETTERS318:83, 1993; Bergers et al., EMBO J. 13:1176, 1994). Human ST2 cDNA andgenomic clones have been isolated as well (Tominaga et al. Biochimica etBiophysica Acta 1171:215, 1992).

Other proteins exhibiting significant sequence homology with IL-1RI aremurine MyD88 (Lord et al., Oncogene 5: 1095-1097, 1990), human rsc786(Nomura et al., DNA Res. 1:27-35, 1994), and a number of Drosophilaproteins, the best characterized of which is Toll (Hashimoto et al.,Cell 52, 269-279, 1988). The tobacco N gene (Whitham et al., Cell78:1101-1115, 1994) is among the additional IL-1R family members.

MyD88, rsc786, Toll, and the tobacco N gene product contain domainsexhibiting significant homology to the cytoplasmic domain of the IL-1RI.The IL-1R AcP and ST2 proteins exhibit sequence similarity to IL-1RI inboth their extracellular and cytoplasmic portions. The B16R protein ofvaccinia virus (Goebel et al., Virology 179:247, 1990) appears to be aviral homolog of IL-1RII.

Identification of additional receptors of this family is desirable. Suchreceptor proteins can be studied to determine whether or not they bindIL-1, and, if so, whether the receptors play a role in mediating signaltransduction. The possible existence of additional affinity-convertingsubunits for receptors of this family can be explored, as well.

SUMMARY OF THE INVENTION

The present invention provides a novel receptor protein designated 2F1.Both soluble and membrane-bound forms of 2F1 are disclosed herein. Thepresent invention also provides isolated DNA encoding 2F1 proteins,expression vectors comprising the isolated DNA, and a method forproducing 2F1 by cultivating host cells transformed with the expressionvectors under conditions appropriate for expression of the 2F1 protein.Antibodies directed against 2F1 are also disclosed. 2F1 finds use ininhibiting prostaglandin synthesis and alleviating inflammation.

DETAILED DESCRIPTION OF THE INVENTION

DNA encoding a novel receptor protein designated 2F1 has been isolatedin accordance with the present invention. Expression vectors comprisingthe 2F1 DNA are provided, as well as methods for producing recombinant2F1 polypeptides by culuring host cells containing the expressionvectors under conditions appropriate for expression of 2F1, thenrecovering the expressed 2F1 protein. Purified 2F1 protein is alsoencompassed by the present invention, including soluble forms of theprotein comprising the extracellular domain.

The present invention also provides 2F1 and immunogenic fragmentsthereof that may be employed as immunogens to generate antibodiesspecific thereto. In one embodiment, the antibodies are monoclonalantibodies.

Human 2F1 clones were isolated as described in example 1. A human 2F1DNA sequence is presented in SEQ ID NO:1, and the amino acid sequenceencoded thereby is presented in SEQ ID NO:2. The protein includes asignal peptide (amino acids −19 to −1) followed by an extracellulardomain (amino acids 1 to 310), a transmembrane region (amino acids 311to 332), and a cytoplasmic domain (amino acids 333 to 522).

Mouse 2F1 cDNA was isolated by cross-species hybridization, as describedin example 2. The DNA and encoded amino acid sequences of this mouse 2F1DNA are presented in SEQ ID NO:3 and SEQ ID NO:4. The protein of SEQ IDNO:4 comprises a signal peptide (amino acids −18 to −1), anextracellular domain (amino acids 1 to 307), a transmembrane region(amino acids 308 to 330), and a cytoplasmic domain (amino acids 331 to519). The mouse and human 2F1 amino acid sequences are 65% identical.

The amino acid sequence of the 2F1 protein indicates that it is a memberof the IL-1 receptor family. Of the known IL-1 receptor family members,2F1 has the highest degree of sequence homology with IL-1R accessoryprotein (IL-1R AcP), T1/ST2, and type I IL-1 receptor (IL-1RI). Themurine 2F1 amino acid sequence of SEQ ID NO:4 is 31% identical to theamino acid sequence of murine IL-1R AcP, 30% identical to that of thefull length murine T1/ST2, and 27% identical to that of the murineIL-1RI. The cytoplasmic domains show slightly greater sequenceconservation (36%-44%) than do the extracellular portions (20%-27%).

The binding assay described in example 3 was conducted to determinewhether 2F1 binds IL-1α, IL-1β, or IL-1 receptor antagonist. Although2F1 is a member of the IL-1 receptor family, it did not bind any of thethree proteins tested.

Human and mouse 2F1 are within the scope of the present invention, asare 2F1 proteins derived from other organisms, including but not limitedto mammalian species such as rat, bovine, porcine, or various non-humanprimates. DNA encoding 2F1 proteins from additional organisms can beidentified by cross-species hybridization techniques. Messenger RNAsisolated from various cell types can be screened in Northern blots todetermine a suitable source of mRNA for use in cloning 2F1 cDNA fromother species.

The term “2F1” as used herein refers to a genus of polypeptides that aresubstantially homologous to a native 2F1 protein (e.g., the protein ofSEQ ID NO:2 or 4), and which exhibit a biological activity of a native2F1 protein. 2F1 proteins of the present invention, includemembrane-bound proteins (comprising an extracellular domain, atransmembrane region, and a cytoplasmic domain) as well as truncatedproteins that retain a desired property. Such truncated proteinsinclude, for example, soluble 2F1 comprising only the extracellulardomain or a fragment thereof. Also included are variants of native 2F1proteins, wherein the variants retain a desired biological activity of anative 2F1. Such variants are described in more detail below.

A 2F1 polypeptide, or fragment or variant thereof, can be tested forbiological activity in any suitable assay. When the cytoplasmic domainis altered (e.g., truncated, or altered by deletion, addition, orsubstitution of amino acid residues), the 2F1 polypeptide can be testedfor biological activity. In a signal transduction assay. Such assaysinclude, but are not limited to, those described in examples 5 to 7below. The altered cytoplasmic domain can be fused to the extracellulardomain of an IL-1 receptor, and the resulting chimeric receptor testedfor the ability to respond to IL-1 by NF-κB activation (see theprocedure in example 5), induction of IL-8 promoter function (example6), or stimulation of prostaglandin E₂ synthesis (example 7). 2F1polypeptides that include an extracellular domain (e.g., soluble 2F1, asdescribed below) can be tested for the ability to inhibit prostaglandinE₂ synthesis in vivo in animal studies. The 2F1 is administered in vivo,and prostaglandin E₂ levels in the animals are measured (before andafter administration of 2F1, and compared to control animals) by anysuitable means, e.g., by ELISA.

One embodiment of the present invention is directed to soluble 2F1polypeptides. Soluble 2F1 polypeptides comprise all or part of theextracellular domain of a native 2F1, but lack the transmembrane regionthat would cause retention of the polypeptide on a cell membrane. Wheninitially synthesized, soluble 2F1 polypeptides advantageously comprisethe native (or a heterologous) signal peptide to promote secretion, butthe signal peptide is cleaved upon secretion of 2F1 from the cell.

One use of soluble 2F1 polypeptides is in blocking a biological activityof 2F1. Soluble 2F1 may be administered to a mammal to bind anyendogenous 2F1 ligand(s), thereby inhibiting the binding of such ligandsto endogenous receptors comprising 2F1. In one embodiment, a soluble 2F1polypeptide is administered to treat pain or inflammation by inhibitingprostaglandin synthesis, as discussed in more detail below.

Soluble 2F1 may be identified (and distinguished from its non-solublemembrane-bound counterparts) by separating intact cells which expressthe desired protein from the culture medium, e.g., by centrifugation,and assaying the medium (supernatant) for the presence of the desiredprotein. The presence of 2F1 in the medium indicates that the proteinwas secreted from the cells and thus is a soluble form of the desiredprotein. Soluble 2F1 may be a naturally-occurring form of this protein.

The use of soluble forms of 2F1 is advantageous for certainapplications. Purification of the proteins from recombinant host cellsis facilitated, since the soluble proteins are secreted from the cells.Further, soluble proteins are generally more suitable for intravenousadministration.

Examples of soluble 2F1 polypeptides include those comprising the entireextracellular domain of a native 2F1 protein. One such polypeptide is asoluble human 2F1 comprising amino acids 1 through 310 of SEQ ID NO:2.Another is a soluble murine 2F1 comprising amino acids 1 through 307 ofSEQ ID NO:4. When initially expressed within a host cell, the solublepolypeptide may additionally comprise one of the heterologous signalpeptides described below that is functional within the host cellsemployed. Alternatively, the polypeptide may comprise the native signalpeptide, such that the 2F1 comprises amino acids −19 through 310 of SEQID NO:2 or amino acids −18 through 307 of SEQ ID NO:4. Soluble 2F1polypeptides include fragments of the extracellular domain that retain adesired biological activity. DNA sequences encoding soluble 2F1polypeptides are encompassed by the present invention.

2F1 fragments, including soluble polypeptides, may be prepared by any ofa number of conventional techniques. A desired DNA sequence may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels.Oligonucleotides that reconstruct the 5′ or 3′ end of a DNA fragment toa desired point may be synthesized. The oligonucleotides may contain arestriction endonuclease cleavage site upstream of the desired codingsequence and position an initiation codon (ATG) at the 5′ terminus ofthe coding sequence. Linkers containing restriction endonucleasecleavage site(s) may be employed to insert the desired DNA fragment intoan expression vector. Alternatively, known mutagenesis techniques may beemployed to insert a stop codon at a desired point, e.g., immediatelydownstream of the codon for the last amino acid of the extracellulardomain.

As a further alternative, the well known polymerase chain reaction (PCR)procedure may be employed to isolate a DNA sequence encoding a desiredprotein, fragment. Oligonucleotides that define the termini of thedesired fragment are employed as primers in the reaction. PCR proceduresare described, for example, in Saiki et al. (Science 239:487, 1988) andin Recombinant DNA Methodology, Wu et al. eds., Academic Press Inc., SanDiego, 1989, pp 189-196.

Regarding the foregoing discussion of signal peptides and the variousdomains of the 2F1 proteins, the skilled artisan will recognize that theabove-described boundaries of such regions of the proteins areapproximate. For example, although computer programs that predict thesite of cleavage of a signal peptide are available, cleavage can occurat sites other than those predicted. Further, it is recognized that aprotein preparation can comprise a mixture of protein molecules havingdifferent N-terminal amino acids, due to cleavage of the signal peptideat more than one site. In addition, the exact boundaries of atransmembrane region may differ from that predicted by a computerprogram. Such forms of 2F1 that retain a desired biological activity areincluded among the 2F1 polypeptides of the present invention.

The present invention provides purified 2F1 polypeptides, bothrecombinant and non-recombinant. Variants and derivatives of native 2F1proteins that retain a desired biological activity are also within thescope of the present invention. 2F1 variants may be obtained bymutations of nucleotide sequences coding for native 2F1 polypeptides. A2F1 variant, as referred to herein, is a polypeptide substantiallyhomologous to a native 2F1, but which has an amino acid sequencedifferent from that of a native 2F1 because of one or more deletions,insertions or substitutions.

The variant amino acid sequence preferably is at least 80% identical toa native 2F1 amino acid sequence, most preferably at least 90%identical. The percent identity may be determined, for example, bycomparing sequence information using the GAP computer program, version6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

DNA encoding such variants is provided by the present invention as well.Such DNA sequences preferably are at least 80% identical to a native 2F1DNA sequence, most preferably at least 90% identical. The percentidentity may be determined using known computer programs, such as theabove-described GAP program.

Alterations of the native amino acid sequence may be accomplished by anyof a number of known techniques. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Methods of making the alterations set forth above aredisclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462.

Variants include conservatively substituted sequences, meaning that oneor more amino acid residues of a native 2F1 is replaced by a differentresidue, but that the conservatively substituted 2F1 polypeptide retainsa desired biological activity of the native protein. Examples ofconservative substitutions include substitution of residues that do notalter the secondary or tertiary structure of the protein.

A given amino acid may be replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other conservative substitutions, e.g., involving substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown.

2F1 proteins also may be modified to create 2F1 derivatives by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of 2F1s may be prepared by linking the chemical moieties tofunctional groups on 2F1 amino acid side chains, or at the N-terminus orC-terminus of an 2F1 polypeptide or the extracellular domain thereof.Other derivatives of 2F1 within the scope of this invention includecovalent or aggregative conjugates of 2F1s with other proteins orpolypeptides, e.g., N-terminal or C-terminal fusions produced byrecombinant DNA technology. For example, the conjugate may comprise aheterologous signal or leader polypeptide sequence at the N-terminus ofa 2F1 polypeptide. The signal or leader peptide co-translationally orpost-translationally directs transfer of the conjugate from its site ofsynthesis to a site inside or outside of the cell membrane or cell wall.

2F1 polypeptide fusions can comprise peptides added to facilitatepurification and identification of 2F1. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(DYKDDDDK) (SEQ ID NO:5), which is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein.Expression systems useful for fusing the Flag® octapeptide to the N— orC-terminus of a given protein are available from Eastman Kodak Co.,Scientific Imaging Systems Division, New Haven, Conn., as are monoclonalantibodies that bind the octapeptide.

The present invention further includes 2F1 polypeptides with or withoutassociated native-pattern glycosylation. 2F1 expressed in yeast ormammalian expression systems may be similar to or significantlydifferent from a native 2F1 polypeptide in molecular weight andglycosylation pattern, depending upon the choice of expression system.Expression of 2F1 polypeptides in bacterial expression systems, such asE. coli, provides non-glycosylated molecules.

N-glycosylation sites in the 2F1 extracellular domain can be modified topreclude glycosylation. N-glycosylation sites in eukaryotic polypeptidesare characterized by an amino acid triplet Asn-X—Y, wherein X is anyamino acid except Pro and Y is Ser or Thr. The human 2F1 proteinextracellular domain contains such triplets at amino acids 72-74, 83-85,131-133, 149-151, 178-180, 184-186, 217-219, and 278-280 of SEQ ID NO:2.The murine 2F1 protein contains such triplets at amino acids 32-34,53-55, 89-91, 93-95, 116-118, 171-173, 176-178, 182-184, 215-217,277-279, and 460-462 of SEQ ID NO:4. Appropriate modifications to thenucleotide sequence encoding this triplet will result in substitutions,additions or deletions that prevent attachment of carbohydrate residuesat the Asn side chain. Alteration of a single nucleotide, chosen so thatAsn is replaced by a different amino acid, for example, is sufficient toinactivate an N-glycosylation site. Known procedures for inactivatingN-glycosylation sites in proteins include those described in U.S. Pat.No. 5,071,972 and EP 276,846.

Additional variants are those in which cysteine residues that are notessential for biological activity are deleted or replaced with otheramino acids, preventing formation of incorrect intramolecular disulfidebridges upon renaturation. As with the other IL-1R family members, theextracellular domain of 2F1 contains three immunoglobulin-like (Ig)domains. Based on alignment of the human 2F1 amino acid sequence withthat of other family members, the cysteines predicted to form thetypical intradomain disulfide bonds of the Ig domains are located atpositions 121, 166, 218, and 279 of SEQ ID NO:2. The first (mostN-terminal) Ig domain includes the first (residue 22) but lacks thesecond cysteine of the pair conserved in other proteins of this family.The first Ig domain of 2F1 thus is predicted to lack the intradomaindisulfide bond that is typical of Ig domains. Like all IL-1R-likeproteins except T1/ST2, mouse and human 2F1 also have a cysteine residuejust a few residues C-terminal to the point of signal peptide cleavage(the cysteine at position 2 of of SEQ ID NO:2 and at position 4 of ofSEQ ID NO:4). 2F1 fragments and variants preferably contain theseconserved cysteines.

Other variants are prepared by modification of adjacent dibasic aminoacid residues to enhance expression in yeast systems in which KEX2protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Human 2F1 contains such KEX2 protease processing sites atamino acids 94-95, 296-297, 345-346, 418-419, and 448-449 of SEQ IDNO:2. Murine 2F1 contains KEX2 protease processing sites at amino acids87-88, 96-97, 231-232, 244-245, 295-296, 339-340, 416-417, 432-433, and446-447 of SEQ ID NO:4. Lys-Lys pairings are considerably lesssusceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg toLys-Lys represents a conservative and preferred approach to inactivatingKEX2 sites.

Naturally occurring 2F1 variants are also encompassed by the presentinvention. Examples of such variants are proteins that result fromalternative mRNA splicing events or from proteolytic cleavage of the 2F1protein, wherein a desired biological activity is retained. Alternativesplicing of mRNA may yield a truncated but biologically active 2F1protein, such as a naturally occurring soluble form of the protein, forexample. Variations attributable to post-translational processing orproteolysis include, for example, differences in the N- or C-terminiupon expression in different types of host cells, due to proteolyticremoval of one or more terminal amino acids from the 2F1 protein(generally from 1-5 terminal amino acids).

2F1 proteins in which differences from the amino acid sequence of SEQ IDNO:2 are attributable to genetic polymorphism (allelic variation amongindividuals producing the protein) are also among the naturallyoccurring variants contemplated herein. The human 2F1 sequence presentedin SEQ ID NO:1 is derived from three cDNA clones from a peripheral bloodlymphocyte library and four PCR clones from the epidermal carcinoma lineKB (see example 1). The codon for alanine 298 is polymorphic, beingpresent in the PBL clones and two of the KB clones, and absent from theother two KB clones. It is also absent from the two mouse clones thatwere derived from an EL4 T cell library. The present invention thusprovides human 2F1 proteins either containing or lacking an alanineresidue at position 298.

The present invention provides isolated DNA sequences encoding the novel2F1 polypeptides disclosed herein. 2F1-encoding DNA encompassed by thepresent invention includes, for example, cDNA, chemically synthesizedDNA, DNA isolated by PCR, genomic DNA, and combinations thereof. Genomic2F1 DNA may be isolated by conventional techniques, e.g., by using theDNA of SEQ ID NOS:1 or 3, or a fragment thereof, as a probe in ahybridization procedure.

Particular embodiments of the present invention are directed to anisolated DNA comprising nucleotides 1 to 1626 of SEQ ID NO:1 (the entirecoding region), nucleotides 58 to 1626 of SEQ ID NO:1 (encoding maturehuman 2F1), nucleotides 381 to 1994 of SEQ ID NO:3 (the entire codingregion), or nucleotides 435 to 1994 of SEQ ID NO:3 (encoding maturemurine 2F1). In other embodiments, isolated DNA sequences encode a 2F1fragment, such as one of the above-described soluble polypeptides. SuchDNAs include a DNA comprising nucleotides 1 to 985 of SEQ ID NO:1 (whichencode amino acids −19 to 310 of SEQ ID NO:2), nucleotides 58 to 985 ofSEQ ID NO:1 (which encode amino acids 1 to 310 of SEQ ID NO:2),nucleotides 381 to 1355 of SEQ ID NO:3 (which encode amino acids −18 to307 of SEQ ID NO:4), and nucleotides 435 to 1355 of SEQ ID NO:3 (whichencode amino acids 1 to 307 of SEQ ID NO:4). DNAs encoding the variousforms of 2F1 disclosed herein, e.g., 2F1 variants and fusion proteins,are encompassed by the present invention.

Nucleic acid sequences within the scope of the present invention includeisolated DNA and RNA sequences that hybridize to the native 2F1nucleotide sequences disclosed herein under moderately or highlystringent conditions, and which encode biologically active 2F1. Moderatestringency hybridization conditions refer to conditions described in,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989).Conditions of moderate stringency, as defined by Sambrook et al.,include use of a prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), hybridization at about 55° C. in 5×SSC overnight, followed bywashing at 50-55° C. in 2×SSC, 0.1% SDS. Highly stringent conditionsinclude higher temperatures of hybridization and washing. The skilledartisan will recognize that the temperature and wash solution saltconcentration may be adjusted as necessary according to factors such asthe length of the probe. In one embodiment, highly stringent conditionsinclude hybridization at 68° C. followed by washing in 0.1×SSC/0.1% SDSat 68° C.

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA sequence may vary from thatpresented in SEQ ID NO:1 or 3, and still encode an 2F1 protein havingthe amino acid sequence of SEQ ID NO:2 or 4, respectively. Such variantDNA sequences may result from silent mutations that occur during PCRamplification, for example. Alternatively, the variant sequence may bethe product of deliberate mutagenesis of a native sequence.

The present invention thus provides isolated DNA sequences encodingbiologically active 2F1, selected from: (a) DNA derived from the codingregion of a native mammalian 2F1 gene (e.g., DNA comprising the codingregion of the nucleotide sequence presented in SEQ ID NO:1 or 3); (b)DNA capable of hybridization to a DNA of (a) under moderately or highlystringent conditions; and (c) DNA which is degenerate as a result of thegenetic code to a DNA defined in (a) or (b). The 2F1 proteins encoded bysuch DNA sequences are encompassed by the present invention.

Examples of 2F1 proteins encoded by DNA that varies from the native DNAsequence of SEQ ID NO:1 or 3, wherein the variant DNA will hybridize tothe native DNA sequence under moderately or highly stringent conditions,include, but are not limited to, 2F1 fragments and 2F1 proteinscomprising inactivated N-glycosylation site(s) or inactivated KEX2protease processing site(s). Further examples are 2F1 proteins encodedby DNA derived from other mammalian species, wherein the DNA willhybridize to the human DNA of SEQ ID NO:1 or the mouse DNA of SEQ IDNO:3.

Purified 2F1 Protein and Uses Thereof

The present invention provides purified 2F1 polypeptides, which may beproduced by recombinant expression systems as described below orpurified from naturally occurring cells. Conventional proteinpurification techniques may be employed.

The desired degree of purity may depend on the intended use of theprotein. A relatively high degree of purity is desired when the proteinis to be administered in vivo, for example. Advantageously, 2F1polypeptides are purified such that no protein bands corresponding toother (non-2F1) proteins are detected by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to 2F1 protein may bedetected by SDS-PAGE, due to differential glycosylation, variations inpost-translational processing, and the like, as discussed above. Mostpreferably, 2F1 is purified to substantial homogeneity, as indicated bya single protein band upon analysis by SDS-PAGE. The protein band may bevisualized by silver staining, Coomassie blue staining, or (if theprotein is radiolabeled) by autoradiography.

One process for producing the 2F1 protein comprises culturing a hostcell transformed with an expression vector comprising a DNA sequencethat encodes 2F1 under conditions such that 2F1 is expressed. The 2F1protein is then recovered from culture medium or cell extracts,depending upon the expression system employed. As the skilled artisanwill recognize, procedures for purifying the recombinant 2F1 will varyaccording to such factors as the type of host cells employed and whetheror not the 2F1 is secreted into the culture medium.

For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be aerylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify 2F1. Some or all of the foregoingpurification steps, in various combinations, can be employed to providea substantially homogeneous recombinant protein.

It is also possible to utilize an affinity column comprising an antibodythat binds 2F1 to purify 2F1 polypeptides by immunoaffinitychromatography. Example 8 describes a procedure for employing the 2F1protein of the present invention as an immunogen to generate monoclonalantibodies.

The foregoing chromatography procedures are among those that may beemployed to purify either recombinant or non-recombinant 2F1.Recombinant cell culture enables the production of the protein free ofthose contaminating proteins that may be normally associated with 2F1 asit is found in nature, e.g., on the surface of certain cell types.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

2F1 preferably is expressed as a secreted polypeptide to simplifypurification. Secreted recombinant polypeptides from a yeast host cellfermentation can be purified by methods analogous to that disclosed byUrdal et al. (J. Chromatog. 206:171, 1984), which includes twosequential, reversed-phase HPLC steps.

Conjugates comprising a 2F1 polypeptide and a detectable agent areprovided herein. The agent preferably is covalently bound to the 2F1polypeptide. Such conjugates find use in in vitro assays, for example.

Suitable agents include, but are not limited to, radionuclides,chromophores, fluorescent compounds, enzymes that catalyze acolorimetric or fluorometric reaction, and the like, with the particularagent being chosen according to the intended application. Suitableradionuclides include, but are not limited to, ¹²³I, ¹³¹I, ^(99m)Tc,¹¹¹In, and ⁷⁶Br. The agents may be attached to the 2F1 using any of theconventional methods by which such compounds are attached topolypeptides in general. Functional groups on amino acid side chains ofan 2F1 may be reacted with functional groups on a desired agent to formcovalent bonds, for example. The agent may be covalently linked to 2F1via an amide bond, hindered disulfide bond, acid-cleavable linkage, andthe like, which are among the linkages that may be chosen according tosuch factors as the structure of the desired agent. Alternatively, the2F1 or the agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for linking variousmolecules to proteins (Pierce Chemical Company, Rockford Ill.). A numberof techniques for radiolabeling proteins are known. Radionuclide metalsmay be attached to 2F1 using a suitable bifunctional chelating agent,examples of which are described in U.S. Pat. Nos. 4,897,255 and4,965,392.

As described in example 7, the signaling (cytoplasmic) domain of 2F1transduces a biological signal that stimulates prostaglandin E₂synthesis. Prostaglandins are naturally occurring long-chain hydroxyfatty acids exhibiting biological effects that include, inter alia,mediating pain and inflammation.

One embodiment of the present invention is directed to the use ofsoluble 2F1 polypeptides to inhibit prostaglandin synthesis. In vivo,signal transduction may be initiated by the the binding of unidentifiedligand(s) to receptors comprising 2F1. Soluble 2F1 is administered to amammal to bind such ligands, thereby inhibiting ligand binding toendogenous cell surface 2F1.

Soluble 2F1 polypeptides may be administered to a mammal to treatconditions that are mediated by a prostaglandin. A condition is saidherein to be mediated by a prostaglandin when the condition is, at leastin part, caused or exacerbated (directly or indirectly) by aprostaglandin.

Such conditions include, but are not limited to, inflammation associatedwith arthritis (especially rheumatoid, arthritis and osteoarthritis),inflammation of the lungs associated with allergy or asthma, adultrespiratory distress syndrome, inflammatory bowel disease, andinflammation resulting from injury (especially injury of a joint). Thedesirability of inhibiting prostaglandins to treat fever, Bartter'ssyndrome, diabetes mellitus, patent ductus arteriosus, and dysmenorrheais discussed in Harrisons's Principles of Internal Medicine, 13thEdition, Vol. 1, Isselbacher et al., Eds., McGraw-Hill, Inc. New York,1994, pp 433-435.

Prostaglandin E₂ (PGE₂) also has been implicated in bone resorption,including the bone resorption associated with rheumatoid arthritis andperiodontal disease (Isselbacher et al., Eds., supra, at page 434). PGE₂has been reported to cause increased vascular permeability, which is anaspect of the inflammatory response that can lead to local edema(Isselbacher et al., Eds., supra, at page 435). Prostaglandins and theirrole in inflammation are discussed further in Pathophysiology: ClinicalConcepts of Disease Processes, 3rd Edition, Price and Wilson, Eds.,McGraw-Hill Book Company, New York, 1986, pp 36-38; and Inflammation:Basic Principles and Clinical Correlates, Second Edition, Galin et al.,Eds., Raven Press, New York, 1992.

Prostaglandins have been suggested to play roles in modulating theimmune response. PGE₂ can suppress mitogen-induced stimulation of humanlymphocytes, for example. Inhibition of PGE₂ thus may be beneficial inpatients in which depressed cellular immunity is attributable, at leastin part, to the action of prostaglandins. (See Isselbacher et al., Eds.,supra, at page 435). Roles for PGE₁ and PGE₂ in angiogenesis have alsobeen suggested.

It is notable that the 2F1 signaling domain transduced a signal thatresulted in activation of the transcription factor NF-κB (see example5). The anti-inflammatory effect of certain drugs (glucocorticoids) isbelieved to be attributable, at least in part, to inhibition of NF-κBactivation (Auphan et al., Science 270:286-290, 1995; Marx, Science270:232-233, 1995). Soluble 2F1 polypeptides thus may be used to inhibitNF-κB activation signals transduced via 2F1.

NF-κB activation has been linked to TNF-induced replication of humanimmunodeficiency virus (HIV) in infected cells, including T cells(Howard et al., Proc. Natl. Acad. Sci. USA 90:2335-2339, 1993). 2F1 isexpressed on T-cells, and an NF-κB activation signal is transduced bythe 2F1 signaling domain. Thus, soluble 2F1 may be employed to reduceHIV expression in HIV-infected cells. An effective amount of soluble 2F1is administered in vivo to inhibit NF-κB activation that results fromsignaling through 2F1. Any HIV replication that would have resulted fromsuch NF-κB activation is thus diminished.

Oligomeric Forms of 2F1

Encompassed by the present invention are oligomers, such as dimers,trimers, or higher oligomers, that contain 2F1. Such oligomers may benaturally occuring or produced by means such as recombinant DNAtechnology.

The 2F1 moieties of the oligomer may be soluble 2F1 polypeptides. Incertain embodiments, the oligomers comprise from two to four 2F1polypeptides.

Oligomers may be formed by disulfide bonds between cysteine residues ondifferent 2F1 polypeptides, or by non-covalent interactions between 2F1polypeptide chains, for example. In other embodiments, oligomerscomprise multiple 2F1 polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the 2F1 polypeptides.Such peptides may be peptide linkers (spacers), or peptides that havethe property of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of 2F1 polypeptides attached thereto, asdescribed in more detail below.

Preparation of fusion proteins comprising heterologous polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fc domain) has been described, e.g., by Ashkenazi et al., (PNAS USA88:10535, 1991); Byrn et al., (Nature 344:677, 1990); and Hollenbaughand Aruffo (“Construction of Immunoglobulin Fusion Proteins”, in CurrentProtocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992). In oneembodiment of the invention, a 2F1 dimer is created by fusing a 2F1 tothe Fc region of an antibody (IgG1). The Fc polypeptide preferably isfused to the C-terminus of a soluble 2F1. A gene fusion encoding the2F1/Fc fusion protein is inserted into an appropriate expression vector.The 2F1/Fc fusion proteins are expressed in host cells transformed withthe recombinant expression vector and allowed to assemble much likeantibody molecules, whereupon interchain disulfide bonds form between Fcpolypeptides to yield divalent 2F1. The desired dimer may be recoveredby conventional procedures, e.g., by affinity chromatography employing aprotein A or protein G column that will bind the Fc moieties.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated, forms of such polypeptides containing the hinge region thatpromotes dimerization are also included. One suitable Fc polypeptide isdescribed in PCT application WO 93/10151, hereby incorporated byreference. This Fc polypeptide is a single chain polypeptide extendingfrom the N-terminal hinge region to the native C-terminus (i.e., is anessentially full length antibody Fc region). Another useful Fcpolypeptide is described in U.S. Pat. No. 5,457,035. The amino acidsequence of the mutein is identical to that of the native Fc sequencepresented in WO 93/10151, except that amino acid 19 has been changedfrom Leu to Ala, amino acid 20 has been changed from Leu to Glu, andamino acid 22 has been changed from Gly to Ala. This mutein Fc exhibitsreduced affinity for Fc receptors. Procedures for preparing a fusionprotein containing an Fc or Fc mutein polypeptide are described furtherin Baum et al. (EMBO J. 13:3992-4001, 1994).

In other embodiments, 2F1 may be substituted for the variable portion ofan antibody heavy or light chain. If fusion proteins are made with bothheavy and light chains of an antibody, it is possible to form an 2F1oligomer with as many as four 2F1 extracellular regions.

Another method for preparing oligomeric 2F1 involves use of a leucinezipper. Leucine zipper domains are peptides that promote oligomerizationof the proteins in which they are found. Originally identified inseveral DNA-binding proteins (Landschulz et al., Science 240:1759,1988), leucine zippers have since been, found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble oligomericproteins are those described in PCT application WO 94/10308. Peptidesthat preferentially form trimers include, for example, the leucinezipper derived from lung surfactant protein D (SPD) described in Hoppeet al. (FEBS Letters 344:191, 1994), and U.S. patent application Ser.No. 08/446,922, hereby incorporated by reference. Recombinant fusionproteins comprising a soluble 2F1 polypeptide fused to a leucine zipperpeptide are expressed in suitable host cells, and the resulting solubleoligomeric 2F1 that forms is recovered from the culture supernatant.

As a further alternative, oligomeric 2F1 may be expressed as arecombinant fusion protein, with or without peptide linkers between the2F1 moieties. Suitable peptide linkers are known in the art, and may beemployed according to conventional techniques. Among the suitablepeptide linkers are those described in U.S. Pat. Nos. 4,751,180 and4,935,233, which are hereby incorporated by reference. A DNA sequenceencoding a desired peptide linker may be inserted between, and in thesame reading frame as, the DNA sequences encoding 2F1, using anysuitable conventional technique. In one embodiment, two soluble 2F1polypeptides are joined by a peptide linker.

The above-described oligomers may be purified by conventional proteinpurification procedures. Immunoaffinity chromatography using an antibodydirected against 2F1 may be employed, for example. Oligomers containingantibody-derived Fc polypeptides may be purified by affinitychromatography, employing a protein A or protein G column that will bindthe Fc moieties.

The present invention provides isolated DNA sequences encoding 2F1polypeptides fused to immunoglobin-derived polypeptides. Such DNAsequences may encode a soluble 2F1 fused to an antibody Fc regionpolypeptide, for example. DNA sequences encoding fusion proteinscomprising multiple 2F1 polypeptide moieties are also encompassed by thepresent invention.

Compositions Comprising 2F1

The present invention provides compositions (including pharmaceuticalcompositions) comprising an effective amount of a purified 2F1polypeptide and a suitable diluent, excipient, or carrier. 2F1polypeptides administered in vivo preferably are in the form of apharmaceutical composition.

The compositions of the present invention may contain a 2F1 protein inany form described herein, including oligomers, variants, derivatives,and biologically active fragments. In one embodiment of the invention,the composition comprises a soluble human 2F1 protein.

2F1 proteins may be formulated according to known methods that are usedto prepare pharmaceutically useful compositions. Components that arecommonly employed in pharmaceutical formulations include those describedin Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack PublishingCompany.

2F1 protein employed in a pharmaceutical composition preferably ispurified such that the 2F1 protein is substantially free of otherproteins of natural or endogenous origin, desirably containing less thanabout 1% by mass of protein contaminants residual of productionprocesses. Such compositions, however, can contain other proteins addedas stabilizers, carriers, excipients or co-therapeutics.

Components of the compositions will be nontoxic to patients at thedosages and concentrations employed. Ordinarily, the preparation of suchcompositions entails combining a mammalian 2F1 polypeptide or derivativethereof with buffers, antioxidants such as ascorbic acid, low molecularweight (less than about 10 residues) peptides, proteins, amino acids,carbohydrates including glucose, sucrose, or dextrans, chelating agentssuch as EDTA, glutathione, or other stabilizers and excipients. Neutralbuffered saline is one appropriate diluent.

For therapeutic use, the compositions are administered in a manner anddosage appropriate to the indication and the patient. Administration maybe by any suitable route, including but not limited to continuousinfusion, local administration, sustained release from implants (gels,membranes, and the like), or intravenous injection.

Antibodies that Specifically Bind 2F1

The 2F1 proteins of the present invention, or immunogenic fragmentsthereof, may be employed in generating antibodies. The present inventionthus provides antibodies that specifically bind 2F1, i.e., theantibodies bind to 2F1 via the antigen-binding sites of the antibody (asopposed to non-specific binding).

Polyclonal and monoclonal antibodies may be prepared by conventionaltechniques. See, for example. Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Kennet et al. (eds.), Plenum Press,New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land(eds,). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1988). Production of monoclonal antibodies that are immunoreactive with2F1 is further illustrated in example 8 below.

Antigen-binding fragments of such antibodies, which may be producedusing conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fab, F(ab′), and F(ab′)₂ fragments. Antibody fragments and derivativesproduced by genetic engineering techniques are also provided.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques, and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993).

Among the uses of the antibodies is use in assays to detect the presenceof 2F1 polypeptides, either in vitro or in vivo. The antibodies findfurther use in purifying 2F1 by immunoaffinity chromatography. Thoseantibodies that additionally can block transduction of a biologicalsignal through 2F1 may be used to inhibit a biological activity mediatedby such signal transduction. Disorders mediated or exacerbated (directlyor indirectly) by signaling through 2F1 are thus treated. A therapeuticmethod involves in vivo administration of an amount of such an antibodythat is effective in inhibiting an undesired 2F1-mediated biologicalactivity. Such antibodies may be administered to inhibit prostaglandinsynthesis, thereby treating one of the above-describedprostaglandin-mediated disorders, for example.

Pharmaceutical compositions comprising an antibody that is directedagainst 2F1, and a suitable, diluent, excipient, or carrier, areprovided herein. Suitable components of such compositions are asdescribed above for compositions containing 2F1 proteins.

Conjugates comprising a diagnostic (detectable) or therapeutic agentattached to the above-described antibodies are provided herein. In oneembodiment, the agent is a radionuclide or drug. Techniques forattaching such agents to antibodies are well known.

Expression Systems

The present invention provides recombinant expression vectors forexpression of 2F1, and host cells transformed with the expressionvectors. Any suitable expression system may be employed. The vectorsinclude an 2F1 DNA sequence operably linked to suitable transcriptionalor translational regulatory nucleotide sequences, such as those derivedfrom a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, an mRNA ribosomal binding site, and appropriate sequenceswhich control transcription and translation initiation and termination.Nucleotide sequences are operably linked when the regulatory sequencefunctionally relates to the 2F1 DNA sequence. Thus, a promoter isoperably linked to an 2F1 DNA sequence if the promoter controls thetranscription of the 2F1 DNA sequence. An origin of replication, whichconfers the ability to replicate in the desired host cells, and aselection gene by which transformants are identified, are generallyincorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnative to the 2F1 gene can be incorporated into expression vectors. Forexample, a DNA sequence for a signal peptide (secretory leader) may befused in frame to the 5′ end of an 2F1 sequence. A signal peptide thatis functional in the intended host cells enhances extracellularsecretion of the 2F1 polypeptide. The signal peptide is cleaved from the2F1 polypeptide upon secretion of 2F1 from the cell.

Suitable host cells for expression of 2F1 polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-treetranslation systems could also be employed to produce 2F1 polypeptidesusing RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, an 2F1 polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant 2F1 polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and an 2F1 DNA sequence areinserted into the pBR322 vector.

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λ P_(L) promoter and a c1857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) andpPLc28 (resident in E. coli RR1 (ATCC 53082)).

2F1 alternatively may be expressed in yeast host cells, preferably fromthe Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast,such as Pichia or Kluyveromyces, may also be employed. Yeast vectorswill often contain an origin of replication sequence from a 2μ yeastplasmid, an autonomously replicating sequence (ARS), a promoter region,sequences for polyadenylation, sequences for transcription termination,and a selectable marker gene.

Suitable promoter sequences for yeast vectors include, among others,promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman etal., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess etal., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.17:4900, 1978), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the 2F1 polypeptide. The α-factor leader sequence is inserted betweenthe promoter sequence and the structural gene sequence. See, e.g.,Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci.USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Other leadersequences suitable for facilitating secretion of recombinantpolypeptides from yeast hosts are known to those of skill in the art. Aleader sequence may be modified near its 3′ end to contain one or morerestriction sites. This will facilitate fusion of the leader sequence tothe structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant 2F1 polypeptides. Baculovirus systems for productionof heterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Established cell lines of mammalianorigin also may be employed. Examples of suitable mammalian host celllines include COS cells derived from monkey kidney cells (e.g., theCOS-1 cell line ATCC CRL 1650, or the COS-7 line ATCC CRL 1651, Gluzmanet al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL163). Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL10) cell lines, and the CV-1/EBNA-1 cell line derived from the Africangreen monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahanet al. (EMBO J. 10:2821, 1991).

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

Expression vectors for use in mammalian host cells can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A usefulsystem for stable high level expression of mammalian cDNAs in C127murine mammary epithelial cells can be constructed substantially asdescribed by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful highexpression vector, PMLSV N1/N4, described by Cosman et al. (Nature312:768, 1984) has been deposited as ATCC 39890. Additional mammalianexpression vectors are pDC406 (McMahan et al., EMBO J. 10:2821, 1991);HAV-EO (Dower et al., J. Immunol. 142:4314, 1989); pDC201 (Sims et al.,Science 241:585, 1988); pDC302 (Mosley et al., Cell, 59:335, 1989); andthose described in U.S. Pat. No. 5,350,683. Other suitable vectors maybe derived from retroviruses.

In place of the native signal sequence, a heterologous signal sequencemay be added, such as the signal sequence for interleukin-7 (IL-7)described in U.S. Pat. No. 4,965,195; the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768(1984); the interleukin-4 receptor signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846.

Nucleic Acids and Uses Thereof

The 2F1-encoding DNAs disclosed herein find use in the production of 2F1polypeptides, as discussed above. DNA and RNA complements of the DNApresented in SEQ ID NOS:1 and 3 are provided herein, along with bothsingle-stranded and double-stranded forms thereof. Fragments of the 2F1nucleotide sequences presented herein are also useful. Such fragmentsdesirably comprise at least about 17 contigous nucleotides of thesequence presented in SEQ ID NO:1 or SEQ ID NO:3, or the complementthereof.

Among the uses of such 2F1 nucleic acids (including fragments) is use asa probe. Such probes may be employed in cross-species hybridizationprocedures to isolate 2F1 DNA from additional mammalian species. As oneexample, a probe corresponding to the extracellular domain of 2F1 may beemployed. The probes also find use in detecting the presence of 2F1nucleic acids in in vitro assays and in such procedures as Northern andSouthern blots. Cell types expressing 2F1 can be identified. Suchprocedures are well known, and the skilled artisan can choose a probe ofsuitable length, depending on the particular intended application. Theprobes may be labeled (e.g., with ³²P) by conventional techniques.

2F1 nucleic acid fragments also find use as primers in polymerase chainreactions (PCR). 5′ and 3′ primers corresponding to the termini of adesired 2F1 DNA may be employed in isolating and amplifying the DNA,using conventional PCR techniques.

Other useful fragments of the 2F1 nucleic acids are antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target 2F1 mRNA (sense) or 2F1DNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of 2F1 cDNA. Such a fragment generally comprises at least about14 nucleotides, preferably from about 14 to about 30 nucleotides. Theability to create an antisense or a sense oligonucleotide based upon acDNA sequence for a given protein is described in, for example. Steinand Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al.,BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. The antisense oligonucleotides thus maybe used to block expression of 2F1 proteins.

Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences. Other examples of sense orantisense oligonucleotides include those oligonucleotides which arecovalently linked to organic moieties, such as those described in WO90/10448, and other moieties that increase affinity of theoligonucleotide for a target nucleic acid sequence, such aspoly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retroviral vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application WO90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

The following examples are provided to illustrate particularembodiments, and not to limit the scope of the invention.

EXAMPLE 1 Isolation of cDNA Encoding Human 2F1

A human 2F1 DNA was isolated by polymerase chain reaction (PCR). Theprimers employed in the reaction were degenerate oligonucleotides basedon two regions within the cytoplasmic domain of the type I IL-1R. Thesetwo regions are among the motifs that are conserved in the IL-1 receptorfamily.

Initially, appropriate conditions for PCR amplification with thedegenerate primers were determined by using human and mouse type I IL-1receptors and mouse T1/ST2 cDNA clones as template. Using the conditionsthat were determined to yield an amplification product from each ofthese cDNAs, PCR was conducted using a 500 kb human yeast artificialchromosome (YAC) as template. This YAC, designated CO2133, contains DNAfrom the human chromosome 2q12 region and is known to include the type IIL-1 receptor, part of the type II IL-1 receptor, and ST2 (Sims et al.,Cytokine 7:483-490, 1995).

The polymerase chain reactions (20 μl) employed 0.5 μl of a 16:1 mixtureof Taq (Perkin-Elmer) and Vent (New England Biolabs) DNA polymerases andcontained 200 pmole of each primer, 200 μM dNTPs and 5-10 μl of humanYAC C02133 DNA, partially purified by extraction from a pulse-field gel.Cycle conditions were: 5 minutes at 94° C., during which time the DNApolymerase mixture was added; 40 cycles of (1 minute at 94° C., 3minutes at 35° C., 1 minute at 72° C.); followed by 10 minutes at 72° C.The reaction products were separated by electrophoresis on a low-meltingtemperature agarose gel. The band containing material between 90 and 150bp in length was excised, melted, and 5 μl used as template in a secondPCR. The second reaction was performed similarly to the first, exceptthat only 20 cycles were run. The reaction products were separated byelectrophoresis on an agarose gel. The 90-150 bp fraction was eluted,and the DNA was rendered blunt-ended using T4 DNA polymerase,phosphorylated using T4 polynucleotide kinase, heated for 10 minutes at65° C., ethanol precipitated, and ligated into a vector designatedpCRScript (Stratagene Cloning Systems, La Jolla, Calif.) in the presenceof restriction enzyme SrfI.

E. coli DH10 cells were transformed with the ligation products. Whitecolonies were picked from Xgal plates, their inserts amplified by PCRusing vector primers, and a small amount spotted on nylon filters. Thefilters were subsequently hybridized at 42° C. in aqueous conditions toa mixture of ³²P-labelled oligonucleotide probes derived from human andmurine type I IL-1R. Filters were washed at 50° C. in 0.3M NaCl. Thehybridization thus was conducted under conditions of relatively lowstringency.

Only 5 out of 180 inserts hybridized. Random DNA sequencing of 9 of thenon-hybridizing inserts revealed that they were derived from yeast DNA.One of the five hybridizing inserts gave a strong hybridization signal,and DNA sequencing revealed it to be amplified from the type I IL-1Rgene. Of the four weakly hybridizing inserts, three came from yeast DNA,and one was found to represent a novel gene, which has been designated2F1.

The thus-isolated 2F1 DNA fragment was used to probe a cDNA libraryprepared from human peripheral blood lymphocytes (PBL), in an effort toisolate a full-length cDNA clone. Hybridizing clones were identified,and three 2F1 cDNA clones were isolated from the PBL library. Fouradditional 2F1 clones were isolated by PCR from the human epidermalcarcinoma line KB (ATCC CCL 17).

A human 2F1 DNA sequence was elucidated by sequencing these clones. Thenucleotide sequence of the coding region is presented in SEQ ID NO:1,and the amino acid sequence encoded thereby is presented in SEQ ID NO:2.The protein of SEQ ID NO:2 is a type I transmembrane protein, with anN-terminal signal peptide (amino acids −19 to −1) followed by anextracellular domain (amino acids 1 to 329), a transmembrane region(amino acids 330 to 351) and a cytoplasmic domain (amino acids 352 to541). The codon for alanine 298 is polymorphic, being present in the PBLclones and two of the KB clones, and absent from the other two KBclones.

EXAMPLE 2 Isolation of Murine 2F1 cDNA

cDNA encoding murine 2F1 was isolated by cross-species hybridization, asfollows. Human 2F1 cDNA was used as a probe to screen a mouse cDNAlibrary derived from the cell line designated EL4 6.1 (MacDonald et al.,J. Immunol. 135:3944, 1985), which is a subclone of the thymoma cellline EL4 (ATCC TIB 39). A hybridizing clone was isolated. The nucleotidesequence of this mouse 2F1 cDNA and the amino acid sequence encodedthereby are presented in SEQ ID NO:3 and SEQ ID NO:4.

The protein of SEQ ID NO:4 comprises a signal peptide (amino acids −18to −1), an extracellular domain (amino acids 1 to 307), a transmembraneregion (amino acids 308 to 330), and a cytoplasmic domain (amino acids331 to 519). The mouse 2F1 amino acid sequence of SEQ ID NO:4 is 65%identical to the human 2F1 amino acid sequence presented in SEQ ID NO:2.

EXAMPLE 3 Binding Assay

2F1 is a member of the IL-1 receptor family, as discussed above. Sincethe 2F1 extracellular domain resembles that of the type I and type IIIL-1 receptors, the ability of 2F1 to bind to IL-1 family members wasinvestigated, as follows.

2F1 was tested for the ability to bind IL-1α and IL-1β (March et al.Nature (Lond.) 315:641, 1985), as well as IL-1 receptor antagonistprotein (Eisenberg et al. Nature 343:341, 1990; Hannum et al., Nature343:336, 1990; and Carter et al., Nature 344:633, 1990). IL-1 receptorantagonist (IL-1ra) binds to IL-1 receptors, but does not transduce asignal. By competing with IL-1 for binding to endogenous IL-1 receptors,IL-1ra inhibits biological effects mediated by IL-1.

A soluble fusion protein designated 2F1/Fc, which comprises the human2F1 extracellular domain joined to the Fc region of a human IgG1, wasgenerated by procedures analogous to those described in Baura et al.(EMBO J. 13:3992-4001, 1994). A soluble type I IL-1R/Fc fusion proteincomprising the extracellular domain of human type I IL-1R fused to theFc region polypeptide was prepared for use as a positive control.

A BIAcore biosensor (Pharmacia Biosensor AB, Piscataway, N.J.) was usedto examine binding of IL-1 ligands to the human 2F1/Fc fusion protein,using procedures essentially as described in Arend et al. (J. Immunol.153:4766-4774, 19. Briefly, a goat anti-human IgG serum directed againstthe Fc region (Jackson Immunoresearch Laboratories, Inc., West Grove,Pa.), covalently coupled to the dextran matrix of a hydrogel chip, wasused to capture the human 2F1/Fc protein. The 2F1/Fc fusion protein thuswas immobilized on the BIAcore chip. The three IL-1 ligands, at severaldifferent concentrations, were reacted with the captured protein, andthe change of mass per unit area over time was measured.

The biosensor analysis demonstrated easily measurable binding of humanIL-1α, IL-1β, and IL-1ra to the human type I IL-1R/Fc fusion protein(positive control). However, no binding of any of the three IL-1proteins to the 2F1/Fc fusion protein was detected. Thus, despite itssignificant sequence homology, 2F1 is not an IL-1 receptor.

EXAMPLE 4 Northern Blot Analysis

The presence of 2F1 mRNA in various cell types was investigated byNorthern blot analysis, using standard techniques. Northern blotspurchased from Clonetech, Palo Alto, Calif., which contain 2 mg of humanpoly A⁺ RNA in each lane, were probed overnight with a ³²P-labeledantisense 2F1 riboprobe at 63° C. in 0.75M NaCl/50% formamide, andwashed at 63° C. in 0.3M NaCl. To ascertain evenness of loading as wellas effectiveness of rRNA removal, the filters were subsequently probedfor GAPDH and 28S rRNA.

A single hybridizing band, migrating with or slightly faster than 28SrRNA, was found in spleen, thymus, leukocyte, liver, lung, heart, smalland large intestine, prostate and placenta. It is possible, butuncertain, that a weak signal was seen in testis and ovary. Nohybridizing band was detected in brain, skeletal muscle, kidney, andpancreas.

EXAMPLE 5 Signaling Assay

The signal transduction capability of the 2F1 cytoplasmic domain wasinvestigated in the following assay. An expression vector encoding achimeric receptor, in which the extracellular and transmembrane portionsof the mouse type I IL-1 receptor (IL-1RI) were fused to the cytoplasmicportion of the human 2F1, was constructed. The vector encoded aminoacids 1 to 362 of the murine IL-1RI fused to amino acids 332 to 522 ofhuman 2F1. In preparing the construct, a BglII site was introduced intothe murine IL-1RI DNA, just 3′ of the transmembrane region. Thisresulted in the valine residue at position 361 of the murine IL-1RIbeing changed to isoleucine, which is the amino acid present in thehuman IL-1RI at that position.

Use of the chimeric receptor made it possible to assay for response to aknown ligand (IL-1). When cells expressing IL-1RI are contacted withIL-1α or IL-1β, a number of responses are induced, including stimulationof nuclear localization of the transcription factor NF-κB (Thanos, D.and T. Maniatis, Cell 80:529-532, 1995). Activated NF-κB complexestranslocate to the nucleus and bind the cognate recognition sequence.

The chimeric receptor was expressed in COS-7 cells (ATCC CRL 1651), andthe ability of IL-1 to activate the transcription factor NF-κB wasexamined in an NF-κB gel assay. A sheep anti-human IL-1RI polyclonalantiserum (designated P3 herein) was used to block the endogenous(cross-reactive) monkey IL-1R, without affecting IL-1 binding to thetransfected murine IL-1RI/2F1 chimera. COS-7 cells transfected with anexpression vector encoding the extracellular and transmembrane portionsof the murine IL-1RI, but no cytoplasmic domain, were employed as anegative control. As a positive control, COS-7 cells were transfectedwith an expression vector encoding full length murine IL-1RI.

The assay procedure was as follows. COS-7 cells were transfected withthe receptor constructs. Two days post-transfection, cells were treatedwith the blocking antibody and stimulated (30 minutes, 1 ng/ml) withhuman IL-1α. Immediately after the blocking and IL-1 stimulation,nuclear extracts were prepared from cell samples, essentially asdescribed by Ostrowski et al. (J. Biol. Chem. 266:12722-33, 1991). Adouble-stranded synthetic oligonucleotide probe containing the κBenhancer element from the immunoglobulin k light chain was 5′ endlabelled by phosphorylation with [γ-³²P]ATP. The nuclear extracts (10μg) were incubated with the ³²P-labeled probe for 20 minutes at roomtemperature, and protein-DNA complexes then were resolved byelectrophoresis in 0.5×TBE 10% polyacrylamide gels (Novex). NF-κBcomplexed with DNA indicates NF-κB activation.

The 2F1 cytoplasmic domain was found to induce NF-κB DNA bindingability, in response to IL-1 stimulation of the chimeric receptormolecule. The induction was comparable in magnitude to that mediated viathe murine IL-1RI (positive control).

EXAMPLE 6 Signaling Assay

The signaling capability of 2F1 was examined further in aninterleukin-8(IL-8) promoter activation assay. When cells expressing thetype I IL-1R are contacted with IL-1, transcription of the IL-8 gene isstimulated (Mukaida et al., J. Biol. Chem. 265:21128-33, 1990). TheIL-1R/2F1 chimeric receptor described in example 5 was employed in thisassay, so that response to a known ligand (IL-1) could be investigated.

A reporter plasmid designated pIL8p, carrying a partial human IL-8promoter fused to the coding region of the human IL-2 receptor alphachain, was prepared. COS7 cells (1×10⁵ cells per well in a 12-welltissue culture plate) were co-transfected with 1500 ng of pIL8p and 500ng of an expression vector encoding the IL-1R/2F1 chimeric receptor.Twenty-four hours post-transfection, the culture medium was changed andthe cells were contacted with a blocking antibody, then stimulated with1 ng/ml human IL-1α or left unstimulated. The blocking antibody was a1:100 dilution of the sheep anti-human IL-1RI polyclonal serum P3 seeexample 5), which at that concentration blocks binding of IL-1 to theendogenous COS7 cell IL-1 receptors, but has no effect on binding ofIL-1 to the mouse IL-1RI portion of the recombinant chimeric receptor.

Twelve to sixteen hours post-stimulation, cells were washed twice withbinding medium containing 5% (w/v) non-fat dry milk (5% MBM), andblocked with 2 ml 5% MBM at room temperature for 30 minutes. Cell werethen incubated at room temperature for 60-90 minutes with 1.5 mls/wellof 5% MBM containing 1 μg/ml of mouse monoclonal antibody 2A3 directedagainst IL-2Rα (Cosman et al., Nature 312:768-771, 1984), with gentlerocking. Cells were washed with 5% MBM, then incubated for one hour atroom temperature with 1 ml/well of 5% MBM containing a 1:100 dilution of[¹²⁵I]goat anti-mouse IgG (Sigma Chemical Company, St. Louis, Mo.).Wells were washed four times with 5% MBM, twice with PBS, then strippedby adding 1 ml 0.5 M NaOH, and the counts per minute determined.

The IL-1R/2F1 chimeric receptor was found to respond to IL-1α byinduction of IL-8 promoter function. Transcription of the reporterconstruct was induced by IL-1 stimulation of the IL-1R/2F1 chimera, toabout half the level mediated by the intact mouse type I IL-1R.

EXAMPLE 7 Prostaglandin Synthesis

In a third assay, the IL-1R/2F1 chimeric receptor was expressed in KBhuman epidermal carcinoma cells (ATCC CCL 17). In the presence ofpolyclonal antiserum that blocks human type I IL-1 receptors (seeexample 5), IL-1 stimulation of the chimeric receptor resulted in thesynthesis of prostaglandin E₂.

EXAMPLE 8 Monoclonal Antibodies Directed Against 2F1

This example illustrates the preparation of monoclonal antibodies thatare immunoreactive with a 2F1 protein. Human 2F1 is expressed inmammalian host cells, such as COS-7 or CV-1/EBNA-1 cells. The expressed2F1 is purified and employed as an immunogen in generating monoclonalantibodies, using conventional techniques such as those described inU.S. Pat. No. 4,411,993. Alternative immunogens include, but are notlimited to, 2F1 fragments (e.g., soluble 2F1 comprising theextracellular domain), a soluble 2F1/Fc fusion protein, or cellsexpressing recombinant 2F1 on the cell surface.

Briefly, mice are immunized with 2F1 emulsified in complete Freund'sadjuvant and injected subcutaneously or intraperitoneally in amountsranging from 10-100 μg. Ten to twelve days later, the immunized animalsare boosted with additional 2F1 emulsified in incomplete Freund'sadjuvant. Mice are boosted thereafter on a weekly to bi-weeklyimmunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot blotassay or ELISA (Enzyme-Linked Immunosorbent Assay), for 2F1 antibodies.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of 2F1 in saline. Three tofour days later, the animals are sacrificed, and spleen cells areharvested and fused to a murine myeloma cell line, e.g., NS1 orpreferably P3×63Ag8.653 (ATCC CRL 1580). Fusions generate hybridomacells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified 2F1 by adaptations of the techniques disclosed in Engvall etal. (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in Beckmann et al. (J. Immunol. 144:4212, 1990). Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-2F1monoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can also be used, as can affinity chromatography based uponbinding to 2F1.

1. A method for determining the presence of a 2F1-binding compound in acomposition, said method comprising: a) contacting a polypeptidecomprising the amino acid sequence of 1 to 310 of SEQ ID NO:2 with acomposition suspected of comprising a 2F1-binding compound; and b)detecting binding of the compound to the polypeptide.
 2. The method ofclaim 1, wherein the polypeptide comprises the amino acid sequence of 1to 332 of SEQ ID NO:2.
 3. The method of claim 2, wherein the polypeptidecomprises the amino acid sequence of 1 to 522 of SEQ ID NO:2.
 4. Themethod of claim 3, wherein the polypeptide comprises the amino acidsequence set forth in SEQ ID NO:2.
 5. The method of claim 1, wherein thepolypeptide is membrane-bound.
 6. The method of claim 5, wherein thepolypeptide is expressed in a host cell comprising an expression vectorencoding the polypeptide comprising the amino acid sequence of 1 to 310of SEQ ID NO:2.
 7. The method of claim 1, wherein the compound comprisesan antigen binding site of an antibody.
 8. The method of claim 7,wherein the compound is an antibody.
 9. The method of claim 8, whereinthe antibody is a monoclonal antibody.